The invention relates to a process for site-directed integration of multiple copies of a gene in a mould, to a transformed mould obtainable by such process, to a process for culturing such transformed mould, and to a process for producing and optionally secreting a desired protein by culturing such transformed mould. In particular, the invention provides a process for preparing a protein by a mould transformed by multicopy integration of at least one expressible gene comprising a structural gene encoding a desired protein into the genome of a mould, especially of moulds belonging to the genus Aspergillus.
In this specification the expression xe2x80x9cexpressible genexe2x80x9d means a structural gene encoding a protein, either homologous or heterologous to the host organism, in combination with DNA sequences for proper transcription and translation of the structural gene, and optionally with secretion signal DNA sequences, which DNA sequences should be functional in the host mould. Further, in this specification the expressions xe2x80x9cmouldxe2x80x9d and xe2x80x9cfilamentous fungusxe2x80x9d are considered as synonyms.
1. Filamentous fungi and especially species such as Aspergillus awamori, Aspergillus niger, Trichoderma reesei and Fusarium graminearum have shown to be attractive hosts for large scale production of homologous and heterologous proteins. They have the capacity to secrete substantial amounts of protein into the medium, large scale fermentation is generally well established and most of them they have a GRAS (Generally Recognized As Safe) status, which makes it possible to use these species in the food and food-processing industry. Moreover, the mould Fusarium graminearum A 3/5, the QuornR myco-protein fungus, has also been used as a commercial human food source in the UK for over 10 years (Royer et al.; Bio/Technology 13 (1995) 1479-1483).
The production of fungal proteins, of either homologous or heterologous origin, by filamentous fungi is usually very efficient and production levels of grams per liter were reached. However, compared to this the production levels of heterologous proteins of mammalian, bacterial or plant origin in moulds are relatively low. In order to improve the production of both homologous and heterologous proteins several strategies have been developed. The basic strategy that is commonly applied to achieve higher protein production in moulds is the introduction of multiple copies of the gene encoding the desired protein.
2. Whereas moulds have been successfully used for the production of enzymes, antibody fragments and peptides at laboratory and commercial scale (xylanase, pectinase, etc), the acceptance of products from these genetically modified organisms (GMO) in the market has experienced some unexpected difficulties in the past few years.
(a) In general there is a growing concern about the use of antibiotic resistance genes in genetically modified organisms. The main reason for this concern is the possibility that such a gene might be transferred into and expressed in gut micro-organisms, which would thereby become antibiotic resistant (xe2x80x9cReport on the use of antibiotic resistance markers in genetically modified food organismsxe2x80x9d published by the Advisory Committee on Novel Foods and Processes, Ministry of Agriculture, Fisheries and Food, England, 1994).
(b) Further, the presence of other foreign DNA such as remnants of vector DNA used in cloning is also undesired.
(c) Another concern is the fact that in general the genetically modified strains contain randomly integrated genetic material. In the perception of some consumer organisations this would constitute an unpredictable safety risk, and could mean a barrier to the acceptation of derived products.
3. Therefore, the recombinant mould should ideally contain multiple copies of the gene encoding the desired protein integrated at only a predetermined locus in the genome and no other foreign DNA should be present in order to produce proteins in moulds in both an economically attractive manner and in a way that deals with the concerns about genetically modified organisms as described above.
The generation of mould strains that meet these criteria has not been reported in literature.
The commonly applied system for integration of single or multiple copies of a gene into the genome of moulds, e.g. Aspergillus, Trichoderma and Fusarium graminearum makes use of plasmids which in addition to the gene encoding a desired protein contain bacterial marker genes encoding resistance to antibiotics (e.g. Ampicillin) and other vector sequences. Therefore, genetically modified moulds will usually contain antibiotic resistance genes and other vector DNA.
Whereas, targeted integrations of a single gene copy have been described regularly (e.g. Timberlake, xe2x80x9cGene Cloning and Analysisxe2x80x9d (Chapter 3) in the book xe2x80x9cMore Gene Manipulations in Fungixe2x80x9d (1991) 51-85, edited by Bennett and Lasure; Gouka et al. Applied and Environmental Microbiology 62 (1996) 1951-1957) it has been proven to be very difficult to obtain mould strains that contain multiple gene copies integrated at a predetermined locus in the genome. Gouka et al. (Curr. Genet. 27 (1995) 536-540) reported the selection of targeted multi-copy integrations at the pyrG locus in A. awamori, but the recombinant strains were obtained from transformations in which DNA was used containing vector sequences and no information was presented on the number of gene copies that were integrated at the pyrG locus. For Aspergillus nidulans a similar observation on targeted tandem integration at the argB locus was published (Van den Hondel and Punt, xe2x80x9cGene transfer systems and vector developmentxe2x80x9d (Chapter 1) in the book xe2x80x9cApplied Molecular Geneticsxe2x80x9d (1991) 1-28, edited by Peberdy et al.).
Several other publications indicate that site-directed integration of multiple gene copies could not be obtained, although it was desired for scientific or commercial purposes, (Kubicek-Pranz et al. J. of Biotech. 20 (1991) 83-94; Van den Hondel et al. Antonie van Leeuwenhoek 61 (1992) 153-160; Verdoes et al. Transgenic Research 2 (1993) 84-92; Archer et al. Antonie van Leeuwenhoek 65 (1994) 245-250; Van Gemeren et al. Applied Microbiology and Biotechnology 45 (1996) 755-763; Van Gemeren, xe2x80x9cExpression and secretion of defined cutinase variants by Aspergillus awamorixe2x80x9d (Chapter 5) Thesis University of Utrecht (1997) ISBN 90-393-1229-X).
4. Previously, two processes have been described in literature that, in principle, might allow the generation of mould strains that contain multiple copies of a gene that are integrated at a predetermined locus in the genome without the presence of other foreign DNA.
The first process describes the preparation of a protein by a fungus transformed by site-directed multicopy integration of an expression vector in the ribosomal DNA locus of the fungal genome as described in International PCT patent application WO-A-91/00920; Unilever, published Jan. 24, 1991. Although the Examples were carried out with yeasts, it was envisaged that such process is also applicable to moulds. Thus such process could make it possible to construct a mould strain in which multiple copies of a gene are integrated at a predetermined locus of the genome, without the presence of other foreign DNA.
However, transformation of moulds follows a somewhat different pattern than the transformation of yeasts. Whereas in the yeast Saccharomyces cerevisiae transforming DNA is integrated into the genome of the cell via homologous recombination at the corresponding homologous site, in filamentous fungi such as the mould Aspergillus awamori DNA integrates mainly via illegitimate recombination at random sites in the genome (Finkelstein, xe2x80x9cTransformationxe2x80x9d Chapter 6 in the book xe2x80x9cBiotechnology of Filamentous Fungixe2x80x9d (1992) 113-156, edited by Finkelstein and Ball). For instance, for the mould A. awamori Gouka et al. (Curr. Genet. 17 (1995) 536-540) performed an analysis on a large number of transformants and showed that DNA integrated via homologous recombination in approximately 10% of the transformants, whereas the remaining 90% integrated randomly. This means that transformants have to be screened for site-directed integration events. Therefore, a process for transformation of moulds as described in WO-A-91/00920 would require lengthy screening procedures because DNA that is introduced into the mould cell can also integrate randomly and not only via homologous recombination at the predetermined site.
The second process describes the site-directed integration of a single gene copy whereby any other heterologous DNA used for cloning and any heterologous mould selection marker are removed, as described in European patent application EP-Al-0 635 574; Gist-brocades N.V., published Jan. 25, 1995.
If this second process would be used for preparing a transformed mould containing multiple gene copies, the process is very cumbersome, because the whole process need be repeated for each subsequent copy that needs to be introduced.
Although the repetition of the second process for obtaining multicopies is mentioned as simple statements in the specification (see e.g. page 3, lines 22-23, page 6, lines 21-25, page 7, lines 29-30, and page 8, lines 9-11, and 15-16), it was not shown in the Examples that it really works. In fact the statement xe2x80x9csequential application of the same technologyxe2x80x9d mentioned on page 8, lines 9-11 confirms the laborious character of this method for introducing multiple gene copies at predetermined loci, covering both a single site and multiple sites.
A further disadvantage of the method described is the risk that the earlier introduced desired foreign DNA is removed during a subsequent repetition of the process.
In summary, items 1-4 above show that there exists a need in the field of mould biotechnology to construct mould strains containing multiple copies of a gene encoding a desired protein that are integrated at a predetermined locus in the genome and that are free of bacterial antibiotic resistance genes or of other foreign DNA such as remnants of vector DNA used in cloning. Ideally, the recombinant microorganism should only contain the heterologous gene encoding the desired protein.
The invention is applicable in the field of mould biotechnology and provides a new and more advanced process for site-directed integration of multiple copies of a gene in a mould without leaving any undesired DNA, i.e. without leaving in the transformed mould the selection marker used for selection of transformants or other DNA used for cloning. The invention is based on the specific introduction of a double-strand break at the chromosomal target in the mould cell which significantly enhances site-directed integration at that locus. Repair of the break with a repair DNA homologous to the regions flanking the break and including multiple copies of at least one gene encoding at least one desired protein will lead to simultaneously integration of those multiple copies at the locus of the break.
The present invention provides a process for transforming a mould, in which
(1) multiple copies of a desired gene are integrated in the chromosome of said mould,
(2) the integration in the mould genome is site-directed via homologous recombination in contrast to the usual random integration of moulds,
(3) such site-directed integration event is selected preferentially over any possible random integration event, e.g. by selecting for the restoration of a defective marker gene,
(4) remaining foreign DNA sequences, e.g. antibiotic resistance genes and DNA originating from other organisms, can be avoided, and
(5) a rare-cutting endonuclease, e.g. I-SceI, is used to introduce a double-strand break in the chromosomal DNA of the mould,
Although the emphasis is given to the use of I-SceI as a rare-cutting endonuclease, it is envisaged that also other rare-cutting endonucleases can be used, including HO Endo-nuclease and VDE, the latter also being known as PI-SceI.